In order to amplify DNA by using PCR, a pair of nucleotide primer, the region between which is a target polynucleotide; a polymerase; and nucleotide triphosphates that are substrates for the polymerase and form the polynucleotide after the amplification, are needed. After the amplification, the target polynucleotide may be detected by any method, for example, by using a specific nucleic acid probe that has a sequence complementary to the target sequence.
In addition, there is a method of amplifying cDNA by using reverse transcription (RT) PCR. In this case, a polymerase having a reverse transcriptional activity may be used.
Moreover, uracil DNA glycosylase (UNG) method, wherein deoxyuridine triphosphate, an unnatural type nucleotide triphosphate, is used to prevent amplification product contamination, is also known (JP3,392,863B).
However, such conventional techniques generally had problems that (1) the number of polymerases that have a reverse transcriptional activity is limited; (2) contamination may not be prevented, because general polymerases often do not incorporate deoxyuridine triphosphate which is a substrate during amplification; and therefore the degradation of uracil N-glycosylase in UNG method cannot be attained; and (3) the higher amplification speed is preferred.
Thus, a method of rapidly amplifying and detecting a long chain target polynucleotide, wherein reverse transcription can be carried out; and deoxyuridine triphosphate can be used, has been demanded.